Methods and compositions for inducing oral tolerance in mammals

ABSTRACT

The present invention relates to methods and pharmaceutical formulations for orally delivering an antigen to induce tolerance. The antigen is combined with derivatized amino acids or salts thereof. The induction of oral tolerance may be applied clinically for the prevention or treatment of auto-immune diseases and clinical allergic hypersensitivities, and for the prevention of allograft rejection.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is based upon two U.S. Provisional PatentApplications: serial No. 60/031,356 filed on Nov. 18, 1996 and serialNo. 60/049,691 filed on Jun. 16, 1997. Applicant claims the benefits ofthe filing dates of the aforesaid provisional applications under 35U.S.C. §119.

FIELD OF THE INVENTION

The present invention relates to methods and compositions useful for theinduction of oral tolerance to a coadministered antigen in mammals.

BACKGROUND OF THE INVENTION

Immunological antibody responses to pathogens are required to preventinfections in the body, whereas, immunological tolerance is a propertyof the immune system that allows for the discrimination of self fromnon-self. A breakdown in immunological tolerance to self antigens allowsthe onset of anti-self immunological responses through the generation ofanti-self antibodies and/or cellular immune responses. This breakdown isresponsible for auto-immune diseases seen in both humans and othermammals.

Allergic immune responses to allergens such as those classicallyobserved in, for example, hay fever, reactions to insect bites andcommon food allergies is suppressed through the generation ofimmunological tolerance to the antigen responsible for the allergy,i.e., the allergen. Repeated exposure to a particular allergen throughcontrolled administration of allergen can induce tolerance in somepatients.

Oral tolerance has been characterized in the literature as a state ofantigen-specific systemic immunological unresponsiveness or tolerance,which is induced by prior oral administration or feeding of antigen. (A.M. Mowat, Immunology Today, Vol. 8, No. 3, 1987, pp. 93-98.) Such astate of systemic hyporesponsiveness to an administered protein orantigen has been observed and reviewed in the art. (H. L. Weiner, Proc.Natl. Acad. Sci. USA, Vol. 91, 1994, pp. 10762-10765; and H. L. Weinerand L. F. Mayer, eds., Annals of NY Acad. Sci., Vol. 778, 1996, pp.xiii-xviii.)

It has been shown that oral co-administration of antigens with choleratoxin B subunit as a delivery agent provides an efficient transmucosaldelivery system for induction of immunological tolerance. (J. B. Sun, etal., Proc. Natl. Acad. Sci. USA, Vol. 91, 1994, pp. 10795-10799; and C.Czerkinsky, et al., Annals NY Acad. Sci., Vol. 778, 1996, pp. 185-193.)In these studies sheep red blood cells (SRBC), horse red blood cells(HRBC) or purified human gamma-globulin (HGG) were used as antigen andcovalently conjugated to the cholera toxin B (CTB) subunit deliveryagent. The SRBC-CTB, HRBC-CTB or HGG-CTB were administered orally tomice to induce oral tolerance to these antigens.

One example of a inflammatory demyelinating autoimmune disease in humansis Multiple Sclerosis. Experimental Autoimmune (a.k.a. Allergic)Encephalomyelitis (EAE) is a paralytic disease of the central nervoussystem (CNS) that can be induced in animals by injection, together withComplete Freund's Adjuvant, of brain or spinal cord homogenate, purifiedMyelin Basic Protein (MBP) or other purified encephalitogenic proteins(derived from brain or spinal cord) or synthetic peptides whose aminoacid sequences resemble those of encephalitogenic components of CNStissues. EAE is widely used as a model for human autoimmune inflammatorydemyelinating disorders such as Multiple Sclerosis (J-B. Sun, et al,Proc. Nat'l Acad. Sci., USA, 93, 7196-7201 (1996)). Certain strains ofanimals display greater susceptibility to the disease, including Lewisrats and SJL/J mice. Cats, dogs, Guinea Pigs and rabbits may also besusceptible. The most common source of active encephalitogens is GuineaPig brain or spinal cord.

The encephalitogen/adjuvant suspension is injected into the footpads ofexperimental animals, inducing the onset of disease symptoms within10-12 days. Prevention or modulation of EAE symptoms has been achievedby induction of oral tolerance via oral administration of large,numerous doses of MBP either before or after induction of the disease.Generally, at least five oral doses are required. Determination ofsynergistic or immune enhancing agents to be administered together withMBP in order to reduce the number or magnitude of the MBP doses requiredto modulate the disease symptoms is desirable. If such agents could beidentified, immunogenic tolerance to these and other types of autoimmunediseases could be promoted.

SUMMARY OF THE INVENTION

The present invention relates to methods and formulations for inducingoral tolerance in a mammal, comprising orally administering to themammal a pharmaceutical formulation comprising an antigen and a deliveryagent or agents comprising at least one derivatized amino acid or a saltthereof in an amount sufficient to induce oral tolerance. These deliveryagents allow the administration of lower or fewer doses of antigen thanare required to induce the same degree of systemic immune suppressionwith the antigen alone. The immune responses involved include, but arenot limited to, systemic antibody production or delayed-typehypersensitivity reactions. In addition, the antigens for use in theinduction of oral tolerance do not have to be covalently linked to thedelivery agents.

It is believed that the foregoing delivery agents, when used in theproportions noted below, enhance the action of the antigens byincreasing the proportion of ingested antigen which reaches the systemiccirculation in its tolerogenic form. It may be that this is achieved bystabilization by the delivery agent of the tolerogenic form or fractionof the antigen in a configuration which may more easily cross themucosal epithelium. It will be understood that the methods andcompositions of the invention are not limited by the foregoing possiblemode of action.

The invention relates to methods of inducing oral tolerance in a mammalwherein the deriviatized amino acid is comprised of an amino acidbearing a free carboxyl group, an amide linkage and a hydrophobic chaincomprised of aromatic and/or aliphatic components.

A preferred embodiment of the invention relates to methods of inducingoral tolerance in a mammal wherein the deriviatized amino acid is anacylated amino acid compound of the formula

Ar is a substituted or unsubstituted phenyl,

R⁵ is C₁ to C₁₀ alkyl, C₁ to C₁₀ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl)phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ to C₁₀ alkyl)naphthyl, (C₁to C₁₀ alkenyl)naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl (C₁ to C₁₀alkenyl), naphthyl (C₁ to C₁₀ alkyl) and naphthyl (C₁ to C₁₀ alkenyl);

R⁵ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁to C₄ alkoxy, —OH, —SH and —CO₂R⁶, cycloalkyl, cycloalkenyl,heteroalkyl, alkaryl, heteroaryl, heteroalkaryl, or any combinationthereof; and

R⁶ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl.

Another preferred embodiment of the invention relates to methods ofinducing oral tolerance in a mammal wherein the deriviatized amino acidis a sulphonated amino acid compound of the formula

Ar—SO₂—(R⁴)—OH  II

wherein Ar and R⁴ are as defined above.

Examples of the aforementioned derivatized amino acids are described inFIG. 1 below and include:

4-[4-(N-salicyloyl)]aminophenyl butyric acid (E352);

N-salicyloyl phenylalanine (E94);

4-[4-(N-benzenesulfonyl)]aminophenyl butyric acid (E198);

3-[4-(N-2,3-dimethoxybenzoyl)]aminophenyl propionic acid (E702);

10-(N-salicyloyl)amino decanoic acid (E597);

4-[4-(N-4 phenylbutyryl)]aminophenyl butyric acid (E445);

4-[4-(N-2 methoxybenzoyl)]aminophenyl butyric acid (E579);

3-[4-(N-2 methoxybenzoyl)]aminophenyl propionic acid (E594); and

4-[4-(N-phenoxyacetyl)]aminophenyl butyric acid (E623).

The present invention also relates to pharmaceutical formulations forinducing oral tolerance in a mammal, comprising an antigen and adelivery agent or agents comprising at least one derivatized amino acidor a salt thereof in an amount sufficient to induce oral tolerance.Preferably, the invention relates to pharmaceutical formulations forinducing oral tolerance, wherein the derivatized amino acid isadministered at a dose of about 100-1000 mg per kg of the subject's bodyweight, preferably at a dose of about 250-750 mg per kg of body weight.

Also contemplated are methods and pharmaceutical preparationsincorporating an adjuvant or adjuvants with the antigen and deliveryagent or agents. The formulations are particularly advantageous forinducing oral tolerance to antigens which otherwise would require largeand/or chronic dosing of antigen to induce such tolerance and which, bythemselves, do not pass or are not taken up in the gastrointestinalmucosa and/or are susceptible to chemical cleavage by acids and enzymesin the gastrointestinal tract. Such antigens include those associatedwith or responsible for the induction of auto-immune diseases, clinical(allergic) hypersensitivities, and allograft rejection, and subunits orextracts therefrom; or recombinantly generated whole proteins, subunitsor fragments thereof; or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of the present invention will be more fully described inconnection with the accompanying drawings, in which:

FIG. 1 provides formulas of preferred derivatized amino acids useful inthe invention.

FIG. 2 is a graphic representation of the titer of serum anti-sheep redblood cell (anti-SRBC) IgG antibodies (determined through the indirectIgG assay) and IgM antibodies (determined through the direct IgM assay)in mice fed SRBC with or without salicyloyl-phenylalanine (E94)derivatized amino acid delivery agent in accordance with Examples 1 and2 and Comparative Examples A, B, C, and D.

FIG. 3 is a graphic representation of the titer of anti-influenzaantibodies (determined through the hemagglutination inhibition assay)present in pooled sera of mice pre-immunized with influenza antigen andfed influenza antigen with or without salicyloyl-phenylalanine (E94)derivatized amino acid delivery agent in accordance with Example 3 andComparative Example E.

FIG. 4 is a graphic representation of the titer of serum anti-HA(influenza) IgG antibodies following single-dose feeding of influenzavaccine A/Johannesburg/39/94 (H3N2) with or withoutN-salicyloyl-4-amino-phenyl butyric acid (E352) derivatized amino aciddelivery agent in accordance with Example 4 and Comparative Example F.

FIG. 5 is a graphic representation of the titer of serum anti-ovalbuminIgG antibodies at week 12 in mice fed two doses of ovalbumin at weeks 0and 4 with or without salicyloyl-phenylalanine (E94) derivatized aminoacid delivery agent and challenged intramuscularly at week 9 withovalbumin and Complete Freund's Adjuvant (CFA) in accordance withExample 5 and Comparative Examples G and I.

FIG. 6 is a graphic representation of the titer of serum anti-ovalbuminIgG antibodies at week 4 in mice fed a single dose of ovalbumin with orwithout salicyloyl-phenylalanine (E94) derivatized amino acid deliveryagent and challenged subcutaneously at week 1 with ovalbumin in CFA inaccordance with Example 6 and Comparative Examples H and I.

FIG. 7 is a graphic representation of of the titer of serum anti-sheepred blood cell (anti-SRBC) IgG antibodies (determined through theindirect IgG assay) on day 14 in mice fed SRBC with or without 3-[4-(N-2methoxybenzoyl)]aminophenyl propionic acid (E594) derivatized amino aciddelivery agent in accordance with Example 7 and Comparative Examples Jand K.

FIG. 8 is a graphic representation of footpad thickness in a DTH assayat day 14 in mice fed a single dose of SRBC with or without4-[4-(N-benzenesulfonyl)]aminophenyl butyric acid (E198) and challengedin the footpad at day 7 in accordance with Examples 8 and 9 andComparative Examples L and M.

FIG. 9 is a graphic representation of footpad thickness in a DTH assayat week 5 in mice fed a single dose of ovalbulmin with or without3-[4-(N-2,3-dimethoxybenzoyl)]aminophenyl propionic acid (E702)derivatized amino acid delivery agent and challenged subcutaneously atweek 3 with ovalbumin in CFA in accordance with Example 10 andComparative Examples N and O.

FIG. 10 is a graphic representation of the Mean Clinical Score for theprogression of EAE over time in Lewis rats fed a single dose of MPB withN-salicyloyl phenylalanine (E94) or3-[4-(N-2,3-dimethoxybenzoyl)]aminophenyl propionic acid (E702)derivatized amino acid delivery agents; MBP alone in 1 dose; or MBPgiven in 5 doses in accordance with Example 11.

FIG. 11 is a graphic representation of the Mean Clinical Score for theprogression of EAE over time in Lewis rats fed a single dose of MPB with4-[4-(N-salicyloyl)]aminophenyl butyric acid (E352) derivatized aminoacid delivery agent; MBP alone in 1 dose; or MBP given in 5 doses inaccordance with Example 11.

FIG. 12 is a graphic representation of the mean Maximal Score per groupand the Mean Disease Index per group (defined as the highest mean scoremultiplied by the duration of symptoms) for Lewis rats dosed with asingle dose of MBP and with 4-[4-(N-salicyloyl)]aminophenyl butyric acid(E352) derivatized amino acid delivery agent; MBP alone in 1 dose; orMBP given in 5 doses in accordance with Example 11. The subscripts inthe group labels refer to the number of paralyzed rats in each group,and the “% protection”, i.e. the percent of animals that were notparalyzed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention uses readily available and inexpensive deliveryagents to provide mammals with oral tolerance to antigens. Oraltolerance is characterized as a state of antigen-specific systemicimmunological hyporesponsiveness induced by the feeding of an antigen.Oral tolerance generally results from large or chronic doses ofantigens. As pointed out hereinabove, the present invention is directedto methods and pharmaceutical formulations comprising an antigen and anderivatized amino acid or salt delivery agent useful to induce oraltolerance to the antigen when the antigen and delivery agent are fedsimultaneously. The delivery agents allow the administration of lower orfewer doses of antigen than are required to induce the same degree ofsystemic immune suppression with the antigen alone. The immune responsesinvolved include, but are not limited to, systemic antibody productionand delayed-type hypersensitivity reactions.

The induction of oral tolerance may be applied clinically for theprevention or treatment of auto-immunie diseases and clinical (allergic)hypersensitivities, and for the prevention of allograft rejection.

Antigens

Antigens suitable for use in the present invention include, but are notlimited to, synthetic or naturally derived proteins and peptides, andparticularly those which by themselves require high doses to induce oraltolerance; carbohydrates including, but not limited to, polysaccharides;lipopolysaccharides; and antigens isolated from biological sources suchas, for example, those associated with or responsible for the inductionof auto-immune diseases, clinical (allergic) hypersensitivities, andallograft rejection and subunits or extracts therefrom; or anycombination thereof.

Special mention is made of antigens associated with the autoimmunediseases of multiple sclerosis, lupus erthymetosis, scleroderma,uveitis, insulin-dependent diabetes mellitus or arthritis. In addition,self-antigens include: nucleic acid; oligodeoxynucleotide;thyroglobulin; thyroid cell surface or cytoplasm; parietal cell; adrenalcell; epidermal cell; uvea cell; basement membrane cell; red cellsurface; platelet cell surface; muscle cell; thymus myeloid cell;mitochondria; secretory duct cell; deoxyribonucleic acid-protein;acetylcholine receptor substance; insulin; central nervous systemantigens such as, myelin basic protein, proteolypid protein, and myelinoligodendrocyte glycoprotein; and other normal hormone and tissuefactors.

Allergens include: benzylpenicilloyl, insulin, ovalbumin, lactalbumin,bermuda grass pollen, timothy grass pollen, orchard grass pollen, andcombinations of grass pollen, ragweed pollen, ragweed antigen E, birchtree pollen, bee venom, snake venom, horse dander, cat epithelial,haddock, house dust mite, Chrysanthemum leucanthemum, Alternari tenuis,trypsin, chymotrypsin, dry rot, baker's yeast, tetanus toxoid,diphtheria toxin, ficin and derivatives thereof.

DELIVERY AGENTS

The delivery agents employed in the practice of the present inventionare derivatized amino acids or salts thereof. The derivatized aminoacids include amino acid amides.

Amino acids which may be used to prepare the delivery agents employed inthe methods and compositions of the invention include any carboxylicacid having at least one free amino group, including both naturallyoccurring and synthetic amino acids. Many amino acids and amino acidesters are readily available from a number of commercial sources such asAldrich Chemical Co. (Milwalkee, Wis., USA); Sigma Chemical Co. (StLouis; Mo., USA); and Fluka Chemical Corp. (Ronkonkoma, N.Y., USA).Methods useful for derivatization of the amino acids identified hereinare disclosed in U.S. Pat. No. 5,958,457, filed May 10, 1995; U.S. Pat.No. 5,709,861, filed Jan. 13, 1995; and PCT/US96/00871, filed Jan. 16,1996, published Jul. 18, 1996 under International Publication NumberWO96/21464.

The preferred naturally occurring amino acids used for derivatzation toproduce the delivery agents used in the methods and compositions hereofin the present invention are alanine, arginine, asparagine, asparticacid, citrulline, cysteine, cystine, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, ornithine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine,hydroxyproline, γ-carboxyglutamate, phenylglycine, or o-phosphoserine.It is particularly desirable to utilize arginine, leucine, lysine,phenylalanine, tyrosine, tryptophan, valine, or phenylglycine assubstrates.

The preferred non-naturally occurring amino acids which may bederivatized for use as delivery agents in the present invention areβ-alanine, α-amino butyric acid, γ-amino butyric acid,γ-(aminophenyl)butyric acid, α-amino isobutyric acid, 6-aminocaproicacid, 7-amino heptanoic acid, β-aspartic acid, aminobenzoic acid,aminophenyl acetic acid, aminophenyl butyric acid, γ-glutamic acid,cysteine (ACM), ε-lysine, ε-lysine, methionine sulfone, norleucine,norvaline, ornithine, d-ornithine, p-nitro-phenylalanine, hydroxyproline, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid andthioproline.

Although the present invention encompasses the use of delivery agentsprepared by derivatization of any of the amino acids discussed above,preferred derivatized amino acids to employ in the methods andformulations of the present invention are those of Formulas I and IIabove. Particularly preferred derivatized amino acids useful as deliveryagents are those referred to above and in FIG. 1 as E352; E94; E198;E702; E597; E445; E579; E594; and E623.

ADJUVANTS

Adjuvants which assist in inducing tolerance include lipopolysaccharides(LPS) and cholera toxin β-subunit.

Pharmaceutical Formulations

Delivery of pharmaceutical formulations comprised of an antigen and adelivery agent (with or without an adjuvant), with the delivery agent oragents described herein are, preferably a derivatized amino acid ofFormulas I and II administered in a dose of about 100-1,000 mg/kg ofbody weight, results in the induction of oral tolerance.

In one embodiment of the present invention, the derivatized amino acidsor salts thereof may be used as delivery agents by simply mixing one ormore derivatized amino acids or salts thereof with the antigen (with orwithout adjuvant) prior to oral administration. In another embodiment,the derivatized amino acids or salts thereof may be used to formmicrospheres or microcapsules containing the antigen (with or withoutadjuvant).

Microspheres containing antigen with or without adjuvant can generallybe of the matrix form or the microcapsule form. The matrix form includesboth a hollow matrix sphere in which the delivery agent forms a matrixshell around a hollow center with the antigen (with or without adjuvant)distributed throughout the matrix and a solid matrix sphere in which thedelivery agent forms a spherical matrix continuum in which the antigen(without or without adjuvant) is distributed. The microcapsule form, onthe other hand, is one in which the encapsulated antigen (either with orwithout adjuvant) is either in solution or in solid form, with thedelivery agent forming a shell around the encapsulated material.

The formulations of the present invention may also include one or moreenzyme inhibitors. Such enzyme inhibitors include, but are not limitedto, compounds such as actinonin, aprotinin or epiactinonin andderivatives thereof. Derivatives of these compounds are disclosed inU.S. Pat. Nos. 5,958,457 and 5,709,861; and in PCT/US96/00871,International Publication Number WO96/21464.

The formulations of the present invention may be formulated into oraldosage units by the addition of one or more excipients, diluents,disintegrants, lubricants, plasticizers, colorants, or dosing vehicles.Preferred oral unit dosage forms include, but are not limited to,tablets, capsules, or liquids. The oral unit dosage forms can includebiologically effective amounts of the antigen (with or without abiologically effective amount of an adjuvant) but can include less thansuch amounts if multiple unit dosage forms are to be used to administera total dosage of the antigen with or without adjuvant. Oral unit dosageforms are prepared by methods conventional in the art.

The delivery agents of the present invention do not alter thephysiological and biological properties of the antigen or the adjuvant.Furthermore encapsulation, if used, need not alter the structure of theantigen.

Inducing Oral Tolerance to Autoimmune Antigens

Also provided herein is a demonstration that the delivery agents of theinvention are capable of promoting suppression of EAE through oraltolerance. EAE is widely used as a model for human autoimmuneinflammatory demyelinating diseases. (See BACKGROUND above.) It isproposed that the derivatized amino acid delivery agents of theinvention act by increasing the fraction of an administered dose of MBPthat is absorbed across the GI epithelia. This is very significant,since tolerance is known to be a highly dose-dependent phenomenon. Thepresence of delivery agents may also lead to a decrease in thevariability in responses that accompanies normal GI absorption. Theinvention thus provides for modulation of immunogenic response, and thusclinical disease, by oral administration of autoantigens accomplished inthe presence of delivery agents using smaller or fewer administereddoses than are required with the antigen alone. It will be understoodthat the methods and compositions of the invention are not limited bythe foregoing possible mode of action.

EXAMPLES

The following examples are intended to illustrate the invention, withoutlimiting its scope.

Example 1 Single Dose Oral Administration of Sheep Red Blood Cells withDelivery Agent

Five female BALB/c mice were fed a single dose suspension of 2.5×10⁹sheep red blood cells (SRBC)+E94 (600 mg/Kg) in Phosphate BufferedSaline (PBS), 0.1 M phosphate and 0.15 M sodium chloride, pH 7.2. Sevendays after completion of oral dosing, mice were primed by footpadinjection of 1×10⁷ SRBC. They were bled on day 14. On day 21 the micewere tested in a Delayed Type Hypersensitivity (DTH) test throughchallenge by injection in the footpad not previously used in primingwith 1×10⁸ SRBC. Footpad thickness was measured before and 24 hoursafter challenge using a Vernier caliper. On day 28 they were bled again.Sera were placed into Eppendorf tubes and assayed for anti-SRBC IgM(days 14 and 28) and anti-SRBC IgG (day 28) by the direct and indirecthemagglutination assays, respectively. (See assay description below.)Prior to assaying, the samples were heat inactivated at 56/C. for 60minutes. Assay data for Example 1 are found in FIG. 2.

Direct Hemagglutination Assay for Anti-SRBC Serum IgM Antibodies

a) Dilute 10μ of sera with 190μ of PBS, pH 7.2 (1/20)

b) On a rigid, U-bottom microtiter plate, mark wells across the rows fordilutions of samples 40; 80; 160; . . . 81,920

c) Place 50μ of PBS in each well

d) In the first well of duplicate rows, add 50μ of heat-inactivated,I/20 diluted sera (first well=1/40) Leave two rows for controls: Mixtureof IgM+IgG positive controls (Rabbit anti-SRBC IgM and Rabbit anti-SRBCIgG), 25μ each diluted I/20 in PBS

e) Serially 2× dilute the sera or controls (50μ) across each row

f) Add 50μ of 0.5% SRBC to each well

g) Cover with pressure-sensitive adhesive plate sealer (COSTAR™)Shakebriefly at low speed to mix

h) Incubate overnight at room temperature, taking care not to disturbthe plate at all

i) Carefully examine for IgM hemagglutination patterns. A positiveresponse appears as a uniform coating of cells adhering to the bottom ofwells. A negative response appears as a tight button of settled cellsthat will stream down if the plate is tilted.

j) Record average of duplicates.

k) To assay for anti-SRBC IgG antibodies DO NOT empty wells. Go on toperform indirect HA assay on the same plate.

Indirect Hemagglutination Assay for Non-agglutinating Anti-SRBC SerumIgG Antibodies

a) In a 15 ml tube, add 11μ of goat anti-mouse IgG (Fc_(y) specific)(2.3 mg/ml) to 5.05 ml PBS (1/460 dilution; approximate amount neededper plate) In a second tube, add 4μ of goat anti-rabbit IgG (F_(c)specific) to 1.8 ml of PBS approximate amount needed (per plate)

b) Add 50μ of diluted goat anti-mouse IgG (FC_(y) specific) to thesample wells (0.25 μg per well) Add 0.25 mg of goat anti-rabbit IgG(F_(c) specific) to each control well

c) Cover with pressure-sensitive adhesive plate sealer (COSTAR™)

d) Shake slowly and briefly to mix

e) Incubate 2 hours at room temp. followed by overnight at 4/C.

f) Carefully examine for IgG hemagglutination titers

g) Record average of duplicates.

Comparative Example A Single Dose Oral Administration of SRBC Alone

Five female BALB/c mice were fed a single dose suspension of SRBC alonewith no delivery agent precisely as described in Example 1. The micewere bled and assayed as described in Example 1. Assay data forComparative Example A are found in FIG. 2.

Example 2 Oral Administration of Sheep Red Blood Cells for FiveConsecutive Days with Delivery Agent

Five female BALB/c mice were fed a suspension of 2.5×10⁹ sheep red bloodcells (SRBC)+E94 (600 mg/Kg) in Phosphate Buffered Saline (PBS) for fiveconsecutive days. The mice were bled and assayed as described inExample 1. Assay data for Comparative Example B are found in FIG. 2.

Comparative Example B Administration of SRBC Alone for Five ConsecutiveDays

Five female BALB/c mice were fed a suspension of SRBC alone with nodelivery agent for five consecutive days as described in Example 2. Themice were bled and assayed as described in Example 1. Assay data forComparative Example B are found in FIG. 2.

Comparative Example C Administration of SRBC Alone for FifteenConsecutive Days

Five female BALB/c mice were fed a suspension of SRBC alone with nodelivery agent for fifteen consecutive days as described in ComparativeExample B. The mice were bled and assayed as described in Example 1.Assay data for Comparative Example B are found in FIG. 2.

Comparative Example D Single Dose Oral Administration of Saline Alone(No SRBC)

Five female BALB/c mice were fed a single oral dose of saline solutionwith no delivery agent as an unfed control. The mice were bled andassayed as described in Example 1. Assay data for Comparative Example Dare found in FIG. 2.

As can be seen in FIG. 2 (Examples 1 and 2; and Comparative Examples A,B, C and D) a single dose of SRBC in the presence of E94 delivery agent(Example 1) suppressed IgM on Day 14 relative to unfed control(Comparative Example D) significantly more than without delivery agent(Comparative Example A), and even lower than 15 doses of SRBC alone(Comparative Example C). On Day 28, IgM for Example 1 (E94+SRBC×1) wasstill lower than that for the Comparative Example D control (90%significance) while no other group was significantly different from theComparative Example D control.

FIG. 2 also shows that for IgG, on Day 28, the Example 1 (E94+SRBC×1)group was lower than the Comparative Example D control (90%significance) while no other groups were significantly different fromthe Comparative Example D control.

Example 3 Oral Administration of Influenza Antigen with Delivery AgentsAfter Priming

Eight OF-1 female mice were primed subcutaneously with a low dose (5 μgper mouse) of vaccine alone (to mimic a non-naive population) on day 0,followed by oral dosing on day 21 with 60 μg of Influenza antigen permouse in solution with 750 mg/kg of E94. Sera were collected every twoweeks, pooled, and assayed for hemagglutination inhibition. (See assaydescription below.) Assay data for Example 3 are found in FIG. 3.

Hemagglutination Inhibition Assay for Anti-HA Antibodies:

A. Hemagglutination Assay to Determine Virus HA Titer

1) Use hard plastic-U-bottom plates.

2) Mark wells 1-10 as 1/10, 1/20 . . . to 1/5120. Mark #12A and 12B ascontrols

3) In a tube, dilute virus suspension 1/10 with PBS.

4) Add 50 μl PBS to each well from #2 to #10 in duplicate rows.

5) Add 100 μl of diluted virus suspension to well #1.

6) Serially 2× dilute 50 μl of the virus across plate until well #10,mixing 7× per dilution.

7) Add 50 μl of well-suspended 0.5% chicken (or sheep) [WHICH ONE?] redblood cells to each well (including control).

8) Cover plate with sealer.

9) Shake briefly on titer plate shaker.

10) Incubate without shaking at room temperature for 30 minutes.

11) Be sure that control wells show a negative pattern (compact settleddrip). If not, wait until they do so.

12) Immediately note the patterns in each well.

13) Record the HA titer of the virus as the highest dilution whichresulted in complete agglutination. If duplicates differ by onedilution, take the average. If they differ by more than one dilution,repeat the assay.

14) This titer provides the dilution of virus suspension which containsone HA unit per 50 μl. Divide this titer by 8 to get the dilution whichwill contain 4 HA units per 0.025 μl for the actual HemagglutinationInhibition (HI) assay.

On the day of the HI assay, prepare just enough diluted virus forback-titration. If the back-titration assay is satisfactory, diluteenough virus for the HI assay of the sera samples.

B. Hemagglutination Inhibition (HI) Assay of Sera

RDE* treat all test sera, reference sera and negative control sera onthe day before the HI assay will be done.

1. RDE* Treatment of Sera to Remove Non-specific Inhibitors

a) Reconstitute RDE (Accurate Chemical and Scientific Corp., Westbury,N.Y.) immediately before use.

b) Add 100 μl serum and 300 μl RDE to 2 ml Eppendorf tube Vortex briefly

c) Incubate at 37/C. overnight

d) Prepare 2.5% sodium citrate solution: 2.5 g sodium citrate plusdistilled water q.s. to 100 ml

e) Add 300 μl sodium citrate solution to the sample tube

f) Incubate at 56/C. for 30 minutes

g) Add 300 μl of PBS. This will result in a 1/10 starting dilution ofserum.

2. HI Assay

A. Back-titration of virus.

a) In a hard plastic plate, add 100 μl of PBS to duplicate wells 1-5(rows A and B) and to two control wells

b) Add 50 μl of the diluted virus suspension (part A #15 above) to well1 of each row

c) Serially 2× dilute across to wells A5 and B5

d) Add 50 μl of 0.5% SRBC to all wells, including the control wells

e) Cover, shake briefly, and incubate 30 minutes

f) The first 3 wells should be completely agglutinated, 4 and 5 shouldbe partially or not at all agglutinated. If this is not the case, thevirus stock should be diluted appropriately to conrect for thisdifference and re-assayed.

B. HI assay:

1. Use flexible plates. Mark columns for duplicate dilution series ofeach test and control serum sample (normal naive serum and positivereference anti-serum). The plate should have 11 columns per row fordilutions plus column 12 for RBC alone.

2. To all wells, except column 1, add 25 μl of PBS.

3. To column 1 wells, add 50 μl of the appropriate RDE-treatment serum(test, positive, or negative control) sample.

4. Serially 2× dilute 25 μl of the sera across to column 11, mixing 7×per dilution.

5. Add 25 μl of diluted virus (containing 4 HA units per 25 μl) to allserum dilution wells, columns 1-11. Add 25 μl of PBS to column 21.

6. Cover plate, mix briefly on shaker, and incubate at r.t. for 30minutes.7.

7. Add 50 μl of well-suspended 0.5% red blood cells to all wells,including the RBC control wells (column 12). Cover and shake briefly.

8. Incubate at room temperature for 30-45 minutes, until RBC controlsshow a compact, negative pattern.

9. Read patterns immediately.

10. Negative control (naive) serum: all wells should show completeagglutination (i.e. no inhibition since there is no anti-HA antibody).

11. Positive control (reference) serum; should see uniform inhibition inthe dilution series up to the theoretical titer of the reference serum.

Comparative Example Single Dose Oral Administration of Influenza AntigenAlone

Eight OF-1 female mice were treated and fed a single dose preparation ofinfluenza antigen alone with no delivery agent as described in Example3. The mice were bled and assayed as described in Example 3. Assay datafor Comparative Example E are found in FIG. 3.

As can be seen in FIG. 3 (Example 3 and comparative Example E) a singledose of influenza antigen with E94 delivery agent (Example 3) suppressedthe production of anti-influenza antibodies relative to control fed onlyinfluenza antigen (Comparative Example E) by more than 2 fold.

Virtually all adult humans have been exposed to Influenza at one or moretimes. Thus, most people have some levels of pre-existing immunity toInfluenza. This preexisting condition will influence theirsusceptibility to infection to cross-reacting strains of the disease. Tosimulate the effects of immunization with oral vaccine in this non-naivepopulation, the mice were pre-immunized with a lower dose of antigenthan would be required to fully immunize them.

Example 4 Oral Administration of Influenza Antigen with Delivery Agents

Ten BALB/c mice were administered a single oral dose of 15 μg ofinfluenza antigen [influenza vaccine A/Johannesburg/39/94 (H3N2)] permouse with 500 mg/kg of E352. Sera were collected every two weeks,pooled, and assayed for anti-hemagglutinin (HA) IgG. (See assaydescription below.) Assay data for Example 4 are found in FIG. 4.

ELISA For Determining Anti-HA IgG Isotype Antibodies in Serum

1. Coat plates with HA antigen, 10 μg/mL in carbonate buffer.

2. Wash×4.

3. Block with Superblock™ or 1/10 diluted Bovine Serum Albumin (BSA).

4. Add 100 μL per well of diluent (Superblock™ or 1/15 diluted BSA) toall except the top row.

5. In the first row, add 150 μL per well of 1/100 diluted test serum induplicate and serially dilute 3× down the plate. Leave 2 empty wells asbackground.

6. Incubate 2 hours at room temperature.

7. Wash×8.

8. To each well, add 100 μl of IgG isotype-specific anti-mousealkaline-phosphatase conjugated antibody (diluted with 4% PEG 6000).20.Incubate overnight at 4/C.

9. Wash×8.

10. Add 100 μL per well of p-nitrophenyl phosphate (pNPP) substratesolution and incubate in the dark with shaking for 30 minutes.

11. Read and record OD₄₀₅ after subtracting the background absorbance.

Comparative Example F Oral Administration of Influenza Antigen Alone

Ten BALB/c mice were fed a single dose preparation of influenza antigen[influenza vaccine A/Johannesburg/39/94 (H3N2)] alone with no deliveryagent as described in Example 4. The mice were bled and assayed asdescribed in Example 4. Assay data for Comparative Example F are foundin FIG. 4.

As can be seen in FIG. 4 (Example 4 and Comparative Example F) a singledoes of influenza antigen with E352 delivery agent (Example 4)significantly suppressed the production of anti-influenza antibodiesrelative to control fed only influenza antigen.

Example 5 Two Dose Oral Administration of Ovalbumin with Delivery Agent

A stock solution was prepared by dissolving Ovalbumin, 10 mg/ml, in 10mM phosphate buffer (pH 7.4). This solution was diluted with buffer toprovide 1.0 mg in a volume of 0.2 ml. Ten BALB/c female mice wereadministered two oral doses of 1 mg ovalbumin per mouse with 600 mg/kgof E94 delivery agent at weeks 0 and 4.

Systemic challenge was achieved by intramuscular (IM) injection of 2mg/ml ovalbumin with 50% Complete Freund's Adjuvant (CFA) at week 9.Serum samples were collected at week 12 and assayed for anti-ovalbumintotal IgG isotypes as described in Example 4 utilizing ovalbumin antigeninstead of HA antigen. Assay data for Example 5 are found in FIG. 5.

Comparative Example G Two Dose Oral Administration of Ovalbumin Alone

Ten BALB/c female mice were fed two oral doses of 1 mg ovalbumin permouse alone with no delivery agent at weeks 0 and 4 as described inExample 5. The mice were challenged, bled and assayed as described inExample 5. Assay data for Comparative Example G are found in FIG. 5.

FIG. 5 illustrates the anti-Ova IgG response in mice immunized orallywith two doses of 1 mg Ovalbumin each with or without delivery agentE94, four weeks apart. They were then challenged intramuscularly withOvalbumin in complete Freund's adjuvant. The response to the challengewith antigen alone (Comp. Ex. G) was the same as in mice given theintramuscular dose alone. However, following feeding in the presence ofdelivery agent (Ex. 5), the response was significantly suppressedcompared to both unfed and antigen-alone fed (Comp. Ex. G) animals. Thisindicates induction of tolerance in the presence of delivery agentfollowing feeding of a dose which is non-tolerizing in the absence ofthe delivery agent.

Example 6 Single Dose Oral Administration of Ovalbumin with DeliveryAgent

A stock solution of ovalbumin was prepared by dissolving Ovalbumin, 125mg/ml, in 10 mM phosphate buffer (pH 7.4). This solution was used toprovide 25 mg in a volume of 0.2 ml. Five BALB/c mice were administereda single oral dose of 25 mg ovalbumin per mouse with 600 mg/kg of E94delivery agent.

Challenge was achieved by subcutaneous (SC) injection of 0.1 mgovalbumin with 50% Complete Freund's Adjuvant (CFA) at week 1. Serumsamples were collected at week 4 and assayed for anti-ovalbumin totalIgG isotypes as described in Example 5. Assay data for Example 6 arefound in FIG. 6.

Comparative Example H Oral Dose Administration of Ovalbumin Alone

Five BALB/c female mice were fed a single oral dose of 25 mg ovalbuminper mouse alone with no delivery agent as described in Example 6. Themice were challenged, bled and assayed as described in Example 6. Assaydata for Comparative Example E are found in FIG. 6.

Comparative Example I Unfed Mice for Control

Five BALB/c female mice were fed nothing for use as a control. The micewere challenged, bled and assayed as described in Example 5 for datadescribed in FIG. 5. The mice were challenged, bled and assayed asdescribed in Example 6 for data described in FIG. 6.

FIG. 6 illustrates that animals fed 25 mg Ovalbumun with delivery agentE94 and then challenged subcutaneously one week later with Ovalbumin incomplete Freund's adjuvant show significantly suppressed serum anti-OvaIgG titers than those which were not pre-fed. While mice fed antigenalone were also suppressed (Comp. Ex. H), this suppression was notstatistically significant, while that induced in the presence of E94(Ex. 6) was significant. Thus, E94 allowed a more consistent suppressionof antibody induction than the antigen alone.

Example 7 Single Dose Oral Administration of Sheep Red Blood Cells withDelivery Agent

Five female BALB/c mice were fed a single dose suspension of 2.5×10⁹sheep red blood cells (SRBC)+E594 (600 mg/Kg) in Phosphate BufferedSaline (PBS), 0.1 M phosphate and 0.15 M sodium chloride, pH 7.2. Sevendays after completion of oral dosing, mice were primed by footpadinjection of 1×10⁷ SRBC. They were bled on day 14. Sera were placed intoEppendorf tubes and assayed for anti-SRBC IgG (day 14) by the indirecthemagglutination assays. (See assay description in Example 1 above.)Prior to assaying, the samples were heat inactivated at 56° C. for 60minutes. Assay data for Example 7 are found in FIG. 7.

Comparative Example J Single Dose Oral Administration of SRBC Alone

Five female BALB/c mice were fed a single dose suspension of SRBC alonewith no delivery agent as described in Example 7. The mice were bled andassayed as described in Example 7. Assay data for Comparative Example Jare found in FIG. 7.

Comparative Example K Single Dose Oral Administration of Saline Alone(No SRBC)

Five female BALB/c mice were fed a single oral dose of saline solutionwith no delivery agent as an unfed control. The mice were bled andassayed as described in Example 7. Assay data for Comparative Example Kare found in FIG. 7.

As can be seen in FIG. 7 (Example 7 and Comparative Examples J and K) asingle dose of SRBC in the presence of E594 delivery agent (Example 7)suppressed IgG on Day 14 relative to unfed control (Comparative ExampleK) significantly more than without delivery agent (Comparative ExampleJ.

Example 8 Single Dose Oral Administration of Sheep Red Blood Cells withDelivery Agent

Five female BALB/c mice were fed a single dose suspension of 2.5×10⁹sheep red blood cells (SRBC)+E594 (600 mg/Kg) as described in Example 7.Seven days after completion of oral dosing, mice were primed by footpadinjection of 1×10⁷ SRBC. On day 14. footpad thickness was measuredaccording to the Delayed Type Hypersensitivity (DTH) method outlined inExample 1. The DTH data for Example 8 are found in FIG. 8.

Example 9 Single Dose Oral Administration of Sheep Red Blood Cells withDelivery Agent

Five female BALB/c mice were fed a single dose Suspension of 2.5×10⁹sheep red blood cells (SRBC)+E198 (600 mg/Kg) and tested for footpadthickness (DTH) as described in Example 8. The DTH data for Example 9are found in FIG. 8.

Comparative Example L Single Dose Oral Administration of Saline Alone(No SRBC)

Five female BALB/c mice were fed a single oral dose of saline solutionwith no delivery agent as an unfed control and tested for footpadthickness (DTH) as described in Example 8. The DTH data for ComparativeExample L are found in FIG. 8.

Comparative Example M Single Dose Oral Administration of SRBC Alone

Five female BALB/c mice were fed a single dose suspension of SRBC alonewith no delivery agent and tested for footpad thickness (DTH) asdescribed in Example 8. The DTH data for Comparative Example M are foundin FIG. 8.

As can be seen in FIG. 8 (Examples 8 and 9 and Comparative Examples Land M) a single dose of SRBC in the presence of E594 delivery agent(Example 8) or E198 delivery agent (Example 9) suppressed the DTHresponse on Day 14 relative to unfed control (Comparative Example L)significantly more than without delivery agent (Comparative Example M).

Example 10 Single Dose Oral Administration of Ovalbumin with DeliveryAgent

A stock solution of ovalbumin was prepared by dissolving Ovalbumin, 10mg/ml in 10 mM phosphate buffer (pH 7.4). This solution was diluted2-fold and used to provide 1.0 mg in a volume of 0.2 ml. Five BALB/cmice were administered a single oral dose of 1.0 mg ovalbumin per mousewith 600 mg/kg of E702 delivery agent.

Challenge was achieved by subcutaneous (SC) injection of 0.1 mgovalbumin with 50% Complete Freund's Adjuvant (CFA) at week 3. DTH assaywas performed as described in Examples 1 and 8. Assay data for Example10 are found in FIG. 9.

Comparative Example N Single Oral Administration of Ovalbumin Alone

Five BALB/c female mice were fed a single oral dose of 1.0 mg ovalbulminper mouse alone with no delivery agent as described in Example 10. Themice were challenged and assayed for DTH as described in Example 10.Assay data for Comparative Example N are found in FIG. 9.

Comparative Example O Unfed Control

Five BALB/c female mice were fed a single oral dose of saline with nodelivery agent as an unfed control. The mice were challenged and assayedfor DTH as described in Example 10. Assay data for Comparative Example Oare found in FIG. 9.

FIG. 9 illustrates that animals fed 1.0 mg Ovalbumun with delivery agentE702 and then challenged subcutaneously 3 weeks later with Ovalbumin incomplete Freund's adjuvant show significantly suppressed response in theDTH than those which were not pre-fed. Mice fed antigen alone at thisdosage were not suppressed (Comparative Example N).

Example 11 Use of Delivery Agents in the MBP/EAE Model for OralTolerance Induction

Groups of five female Lewis rats were given one or five oral doses ofMyelin Basic Protein (MBP, 1.0 mg per dose every 2-3 days) or a singleoral dose of 1.0 mg of MBP together with delivery agents E94, E352 orE702. Two days after the (last) oral dose, all groups were challenged inthe footpad with 0.05 mg of MBP emulsified with Complete Freund'sAdjuvant containing Mycobacterium tuberculosis H37Ra, 4 mg/ml. Clinicalsigns of disease were observed starting on Day 11 after the challengeand rated on a scale of 0 (no disease) to 5 (death). Data for Example 11are provided in FIGS. 10, 11 and 12.

FIGS. 10 and 11 show the suppression of clinical disease by a singledose of MPB with E94 and E702 (FIG. 10) and E352 (FIG. 11). The presenceof delivery agents suppressed disease symptoms to a degree statisticallyidentical to 5 doses of MBP alone, and significantly more than a singledose of MBP alone at the time points indicated. In addition, the meanday of onset of paralysis (defined as a clinical disease score less thanor equal to 1) was delayed from Day 13 after a single dose of MBP aloneto Day 15 in the presence of E94 or E702 and Day 16 in the presence ofE352.

FIG. 12 shows the mean Maximal Score per group and the Mean DiseaseIndex per group (defined as the highest mean score multiplied by theduration of symptoms) for rats dosed with a single dose of MBP and E352vs. one or five doses of MBP alone. In both cases, the presence of E352suppressed these disease parameters significantly compared with the doseof MBP alone, and was statistically identical to the five-dose MBPgroup.

The subscripts in the group labels refer to the number of paralyzed ratsin each group, and the “% protection”, i.e. the percent of animals thatwere not paralyzed.

We claim:
 1. A method of inducing oral tolerance in an animal,comprising orally administering to said animal a pharmaceuticalformulation comprising an antigen and a delivery agent comprising atleast one derivatized amino acid or a salt thereof in an amountsufficient to induce tolerance to said antigen.
 2. The method of claim1, wherein the derivatized amino acid is an acylated amino acid compoundof the formula

wherein: Ar is a substituted or unsubstituted phenyl,

R¹ is C₁ to C₁₀ alkyl, C₁ to C₁₀ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl)phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ to C₁₀ alkyl)naphthyl, (C₁to C₁₀ alkenyl)naphthyl; R¹ is optionally substituted with C₁ to C₄alkyl, C₁ to C₄ alkenyl, C₁ to C₄ alkoxy, —OH, —SH and —CO₂R²,cycloalkyl, cycloalkenyl, heteroalkyl, alkaryl, heteroaryl,heteroalkaryl, or any combination thereof; and R² is hydrogen, C₁ to C₄alkyl or C₁ to C₄ alkenyl.
 3. The method of claim 1, wherein thederivatized amino acid is a sulphonated amino acid compound of theformula Ar—SO₂—(R)—OH  (II) wherein: Ar is a substituted orunsubstituted phenyl,

R¹ is C₁ to C₁₀ alkyl, C₁ to C₁₀ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl)phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ to C₁₀ alkyl)naphthyl, (C₁to C₁₀ alkenyl)naphthyl; R¹ is optionally substituted with C₁ to C₄alkyl, C₁ to C₄ alkenyl, C₁ to C₄ alkoxy, —OH, —SH and —CO₂R²,cycloalkyl, cycloalkenyl, heteroalkyl, alkaryl, heteroaryl,heteroalkaryl, or any combination thereof; and R² is hydrogen, C₁ to C₄alkyl or C₁ to C₄ alkenyl.
 4. The method of claim 1, wherein the antigenis selected from the group consisting of synthetic proteins, naturallyproduced proteins, synthetic peptides, naturally produced peptides,carbohydrates and lipopolysaccharides.
 5. The method of claim 1, whereinthe antigen is associated with the induction of auto-immune diseases,clinical (allergic) hypersensitivities or allograft rejection, andsubunits or extracts therefrom.
 6. The method of claim 1, wherein theformulation incorporates the derivatized amino acid at a dose of100-1000 mg per kg of body weight.
 7. The method of claim 6, wherein theformulation incorporates the derivatized amino acid at a dose of 250-750mg per kg of body weight.
 8. The method of claim 1, wherein theformulation additionally incorporates an enzyme inhibitor.
 9. The methodof claim 1, wherein the formulation additionally incorporates anadjuvant.
 10. A pharmaceutical formulation for inducing oral tolerancein a mammal, comprising an antigen and a delivery agent comprising atleast one derivatized amino acid or a salt thereof in an amountsufficient to induce oral tolerance.
 11. The pharmaceutical formulationof claim 10, wherein the derivatized amino acid is an acylated aminoacid compound of the formula

wherein: Ar is a substituted or unsubstituted phenyl

R¹ is C₁ to C₁₀ alkyl, C₁ to C₁₀ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl)phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ to C₁₀ alkyl)napthyl, (C₁to C₁₀ alkenyl)naphthyl; R¹ is optionally substituted with C₁ to C₄alkyl, C₁ to C₄ alkenyl,C₁ to C₄ alkoxy, —OH, —SH and —CO₂R²,cycloalkyl, cycloalkenyl, heteroalkyl, alkaryl, heteroaryl,heteroalkaryl, or any combination thereof; and R² is hydrogen, C₁ to C₄alkyl or C₁ to C₄ alkenyl.
 12. The pharmaceutical formulation of claim10, wherein the derivatized amino acid is a sulphonated amino acidcompound of the formula Ar—SO₂—(R)—OH  II wherein: Ar is a substitutedor unsubstituted phenyl

R¹ is C₁ to C₁₀ alkyl, C₁ to C₁₀ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl)phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ to C₁₀ alkyl)naphthyl, (C₁to C₁₀ , alkenyl)naphthyl; R¹ is optionally substituted with C₁ to C₄alkyl, C₁ to C₄ alkenyl,C₁ to C₄ alkoxy, —OH, —SH and —CO₂R²,cycloalkyl, cycloalkenyl, heteroalkyl, alkaryl, heteroaryl,heteroalkaryl, or any combination thereof; and R² is hydrogen, C₁ to C₄alkyl or C₁ to C₄ alkenyl.
 13. The pharmaceutical formulation claim 10,wherein the derivatized amino acid is administered at a dose of about100-1000 mg per kg of body weight.
 14. The pharmaceutical formulationclaim 10, wherein the derivatized amino acid is administered at a doseof about 250-750 mg per kg of body weight.
 15. The pharmaceuticalformulation of claim 10, wherein the antigen is selected from the groupconsisting of synthetic proteins, naturally produced proteins, syntheticpeptides, naturally produced peptides, carbohydrates andlipopolysaccharides.
 16. The pharmaceutical formulation of claim 10,wherein the antigen is associated with the induction of auto-immunediseases, clinical (allergic) hypersensitivities or allograft rejection,and subunits or extracts therefrom.
 17. The pharmaceutical formulationof claim 10, wherein the formulation additionally incorporates anadjuvant.
 18. The pharmaceutical formulation of claim 10, wherein theformulation additionally incorporates an enzyme inhibitor.
 19. Themethod of claim 2, wherein the derivatized amino acid is an acylatedamino acid compound of the formula


20. The method of claim 2, wherein the derivatized amino acid is anacylated amino acid compound of the formula


21. The method of claim 2, wherein the derivatized amino acid is anacylated amino acid compound of the formula


22. The method of claim 2, wherein the derivatized amino acid is anacylated amino acid compound of the formula


23. The method of claim 2, wherein the derivatized amino acid is anacylated amino acid compound of the formula


24. The method of claim 2, wherein the derivatized amino acid is anacylated amino acid compound of the formula


25. The method of claim 2, wherein the derivatized amino acid is anacylated amino acid compound of the formula


26. The method of claim 2, wherein the derivatized amino acid is anacylated amino acid compound of the formula


27. The method of claim 2, wherein the derivatized amino acid is anacylated amino acid compound of the formula